JP5301565B2 - Biomaterial manufacturing method - Google Patents

Description

  The present invention relates to a biomaterial and a method for producing the same, and more particularly to a method for producing a biomaterial from decellularized fish scales. The manufactured biomaterial can be used in the field of tissue repair and transplantation.

  Tissue engineering is closely related to biological and pharmaceutical biomaterials, and can enhance angiogenesis, tissue integration, and tissue remodeling in patients undergoing transplant surgery. Biomaterials are artificially synthesized materials that are applied to the reconstruction, repair or repair of prosthetic organs and thus replace human tissue.

  For decades, collagen fibers, hydroxyapatite (HAP) and tricalcium phosphate (TCP) are all biomaterials that are proven by many data and can be safely used for human tissue transplantation. However, these biomaterials have drawbacks such as insufficient mechanical strength, residual chemical substances in the cross-linking process, and zoonotic disease infection.

  Accordingly, there is a need to provide new biomaterials with enhanced mechanical strength and reduced probability of zoonotic infection during the process of tissue repair and transplantation.

  It is an object of the present invention to provide a new biomaterial with enhanced mechanical strength and reduced probability of zoonotic infection during the process of tissue repair and transplantation.

  Yet another object of the present invention is to provide the biomaterial described above and use it for tissue repair and tissue transplantation.

  Still another object of the present invention is to provide a method for producing the biomaterial described above.

  The present invention provides a method for producing biomaterials from fish scales, the method comprising the steps of decellularizing the fish scales and further crushing them into a certain amount of granules, these granules comprising a spongy matrix and a spongy powder. And mixtures with.

  The method for producing the biomaterial of the present invention comprises extruding the spongy matrix and the spongy powder at a temperature of 200 ° C. or lower to form a flaky form or a lump form, or You may further include the step which makes it a specific shape using a specific metal mold | die.

  The method for producing a biomaterial of the present invention may further include a step of dewatering the fish scales before crushing the fish scales. In the dehydration step, the fish scales may be dehydrated until the water content is less than 50%. A step of washing the fish scales may be further included before the step of dewatering the fish scales. However, the step of decellularizing the fish scales may be before the step of washing the fish scales. The step of decellularizing the fish scales may be before the step of dehydrating the fish scales.

  The method for producing the biomaterial of the present invention may further comprise separating the spongy matrix and the spongy powder from the granules.

  As the raw fish scales in the present invention, fish scales having an average diameter of less than 20 cm, for example, fish scales having a large size (10 to 20 cm) are possible, but fish scales having a small size may be used. In the method for producing the biomaterial of the present invention, there is an emphasis on retaining the natural bond and the three-dimensional structure of the fish scale biomaterial, and the degree of fish scale crushing is not particularly limited. Generally, fish scales are ground until the average size of the granule diameter is less than 10,000 μm. When the average size of the fish scale material to be used is less than 10,000 μm and it is necessary to further pulverize appropriately, it is pulverized to a smaller granule size. For example, it is possible to grind until the average size of the granule diameter is less than 10 μm.

  The present invention provides still another method for producing a biomaterial from fish scales. The method includes the steps of decellularizing fish scales and further performing an extrusion process to obtain a specific shape using a flake shape or an irregular shape, or a specific mold.

  In the method for producing a biomaterial of the present invention, the extrusion step is performed at a temperature of 200 ° C. or lower. However, the step of washing the fish scales may be further included before the fish scale extrusion process. It may further include a step of dewatering the fish scales after the end of the washing step and before performing the fish scale extrusion process. In the dehydration step, the fish scales may be dehydrated until the water content is less than 50%. However, the step of decellularizing fish scales may be before the step of washing fish scales. The step of decellularizing the fish scales may be before the step of dehydrating the fish scales. A step of immersing fish scales in water may be further included before performing the fish scale extrusion process.

  According to a specific method for producing a biomaterial from the fish scales of the present invention, the production process includes the steps of dehydrating the decellularized fish scales until the water content is less than 50%, and the dehydrated fish scales, Crushing until the average diameter of the milled granules is less than 10,000 μm, each milled granule comprising a mixture of a spongy matrix and a spongy powder. In one embodiment of the present invention, fish scales are dehydrated until the water content is less than 25%, and the average size of the diameter of the crushed granules is less than 5,000 μm.

  On the other hand, the process for producing a biomaterial from the fish scales of the present invention includes performing the treatment at a temperature of 200 ° C. or lower.

  The present invention further provides a biomaterial produced from fish scales using the method.

  The biomaterial produced using the method of the present invention retains the original binding and three-dimensional structure of the fish scale biomaterial, which has good mechanical strength and is used for tissue repair and tissue transplantation. And it is possible to greatly reduce the probability of zoonotic infection in the process of tissue repair and transplantation.

Schematic of the first flow of the method for producing the biomaterial of the present invention Schematic of the second flow of the method for producing the biomaterial of the present invention Schematic of the first flow of the method for producing biomaterials of Example 1 and Example 2 of the present invention Schematic of the second flow of the method for producing biomaterials of Example 1 and Example 2 of the present invention Schematic of the first flow of the method for producing the biomaterial of Example 3 of the present invention Schematic of the second flow of the method for producing the biomaterial of Example 3 of the present invention SEM (Scanning Electron Microscope) diagram of 3T3 cells (fibroblasts) cultured for 5 days with the biomaterial of the present invention SEM diagram of osteoblasts cultured for 5 days with the biomaterial of the present invention Confocal micrograph of 3T3 cells (fibroblasts) cultured with the biomaterial of the present invention for 5 days Confocal micrograph of osteoblasts cultured for 5 days with the biomaterial of the present invention Hematoxylin and eosin (H & E) staining diagram of osteoblasts cultured for 5 days with the biomaterial of the present invention

  Hereinafter, in order to further disclose the advantages and ideas of the present invention, it will be described in detail with reference to the drawings.

  Reference is made to FIG. 1A, which is a schematic diagram of a first flow of a method for producing a biomaterial of the present invention. The biomaterial manufacturing process of the present invention includes the steps of decellularizing the fish scale 9 (step S10) and dehydrating the fish scale 9 until the water content is less than 50% (step S20). The average size of the granules of fish scale 9 after dehydration and pulverization is less than 10,000 μm. Preferably, the fish scale 9 is dehydrated until the water content is less than 25%, and pulverized until the average size of the granule diameter is less than 5,000 μm.

  The fish scales 9 are provided for the production of biomaterials in a refrigerated or frozen state. The present invention selects fish scales 9 having an average diameter of less than 20 cm. First, the fish scale 9 is decellularized (step S10). Decellularization is performed on fish scale 9 using reagents such as hypotonic solution, surfactant, Triton X-100, sodium dodecyl sulfate (SDS), proteolytic enzyme inhibitor, deoxyribonuclease (DNase), and ribonuclease (RNase). Remove attached cells. The purpose of this is that biomaterials produced from fish scale 9 can affect tissue repair and transplantation by cells attached to the surface, and thus induce an immune response in the recipient, and can also cause rejection. Is to avoid. Before performing the dewatering operation of the fish scale 9 (step S12), it is first necessary to wash the fish scale 9 using another detergent (step S11). These detergents can include, but are not limited to, surfactants, detergents, hot water, and polar solvents such as 60 ° C ethanol. However, the present invention does not limit any specific washing step, but in any case, it is necessary to wash fish scale 9 to such an extent that it passes the limulus test (limulus amebocyte lysate, LAL test). The test value in the test must be less than 1,000 Eu / ml.

  Reference is made to FIG. 1B, which is a schematic diagram of a second flow of the method for producing a biomaterial of the present invention. The steps are generally the same as the first flow, except that the decellularization step (step S10) is located after the washing step (step S11). Regardless of whether the decellularization operation (step S10) is performed before the fish scale 9 is washed (step S11), or the decellularization operation (step S10) is performed after the fish scale 9 is washed (step S11). Can proceed to the following steps in sequence.

  Thereafter, the fish scale 9 is dehydrated (step S12). The dewatering method can be a compressed air dehydrator (air spray), oven, freeze drying or other conventional dewatering methods. Further, the fish scale 9 may be dried by immersing the fish scale 9 in ethanol or another polar organic solvent. This is to dehydrate the fish scales 9 until the water content is less than 50%, and in a preferred state, the water content is less than 25%. Subsequently, these dewatered fish scales 9 are pulverized until the average size of the diameter of the granules is less than 10,000 μm. In a preferred state, the diameter of the granules is about 5,000 μm. Further, the pulverized granule contains a mixture of the spongy matrix 14 and the spongy powder 15. Therefore, the present invention further separates the spongy matrix and the spongy powder by filtering the pulverized granules using a filter device such as “sieving”. In this filtering step, vibration means may be used to enhance the sieving effect. Thereby, the granules filtered by the filter device are used as a biomaterial in the form of a powder, and the granules remaining after the filtering are used as a biomaterial in the form of a matrix. Therefore, the apparatus which grinds the fish scale 9 is not limited to a specific grinder. If the average size of the granules after pulverization does not exceed 10,000 μm, a preferable state may be any pulverizer that reduces the size of fish scale 9 as long as the diameter is less than 5,000 μm.

  In order to produce sterile biomaterials, the manufacturing process may further include performing extrusion (step S16), filtering, whole or partial drying, and sterilization. In a preferred embodiment, these steps can be performed whether or not under heating conditions. The biomaterial may be mixed with various connective tissue repair compositions and other active / inactive ingredients.

  Further, by extruding the fish scales 9 after dehydration, it is possible to produce a biomaterial having a specific shape using a flake shape, an indeterminate shape, or a specific mold. This extrusion process may include a process of temperature change (heating, low temperature and thus freezing), but it should not be 200 ° C. or higher. After the washing step (step S11), the fish scale 9 is placed at a temperature of 200 ° C. or lower and extruded (step S16) to obtain a specific shape 17 using a flake shape, an irregular lump shape, or a specific mold. Manufactured biomaterials. The fish scale 9 after dehydration is easy to extrude. Before performing an extrusion process (step S16), you may add arbitrarily the step (step S18) which immerses the fish scale 9 in water. However, this step needs to be performed after the cleaning step (step S11). Further, the present invention has yet another embodiment, regardless of whether the biomaterial is in the form of a matrix or powder, and any of the extruding steps (step S16) can be performed, The biomaterial is converted into Form 17 using a flake shape, an irregular shape, or a specific mold. The extrusion process is performed at a temperature of 200 ° C. or lower. However, the heat treatment of the present invention is not limited to the extrusion process as described above, and those skilled in the art can use other heat treatment methods such as various types of heat extrusion, heat pressurization, and molding steps. It is also possible to manufacture a biomaterial having a specific shape using the flake shape, the irregular lump shape, or a specific mold.

  Since the biomaterial of the present invention contains a tissue repair factor, it is possible to produce a tissue repair material. This tissue repair material is used to repair various tissue injuries and tissue defect sites. For example, the biomaterial of the present invention can be prepared as an injection preparation for a bone defect site, cartilage repair site, alveolar repair site or other soft tissue defect site or the vicinity thereof. In another embodiment, the biomaterial is a coating material for transplantation or implantation at or near a bone defect site, cartilage repair site or other tissue defect site in a surgical operation. Therefore, the present invention can be used to obtain a connective tissue for surgical transplantation having a tissue repair material from fish scales, particularly for connective tissue defect sites.

  As described above, the present invention relates to a biomaterial manufactured from fish scale 9, and the biomaterial is a powder; a matrix; or a flake-shaped or irregular-shaped lump, or a material having a specific shape by a specific mold. Thus, it can be applied to various tissue repair and transplantation fields. Regardless of whether the material obtained from these fish scales 9 is a powder or a matrix, a flaky shape or an indeterminate shape, or a biomaterial made into a specific shape using a specific mold, any tissue repair is possible. There are factors and regularity may be different (the repair method may be different depending on the shape. For example, the repair method is different between bone repair and skin repair).

  The technical contents of the present invention are described in detail below, but the present invention is not limited to these.

Example 1: Matrix Form Reference is made to FIG. 1C, which is a schematic diagram of a first flow for producing the biomaterial of Example 1 of the present invention. This process starts from the step of decellularizing fish scales (step S20). Subsequently, a fish scale cleaning operation (step S19) is performed. The test value in the fish scale limulus test after washing should be less than 1,000 Eu / ml. Reference is made to FIG. 1D, which is a schematic diagram of a second flow for producing the biomaterial of Example 1 of the present invention. This process starts from a fish scale washing operation (step S19). The test value in the fish scale limulus test after washing should be less than 1,000 Eu / ml. Subsequently, a step of decellularizing the fish scale (step S20) is performed.

  Regardless of whether the decellularization operation (step S20) is performed before washing the fish scales (step S19) or the decellularization operation (step S20) is performed after the fish scales are washed (step S19), Either can proceed to the next step in sequence.

  Then, it progresses to step S21 and fish scales are dehydrated until the water content becomes less than 50%. Next, in step S22, the dehydrated fish scales are pulverized until the average size of the diameter of the granules is less than 10,000 μm. These granules contain a mixture of a spongy matrix and a spongy powder. Step S23 is a step of performing a filtering process, and the matrix is separated from the mixture using a filter device.

  Thus, a matrix is obtained after the filtering step, which includes fibrous tissue composed of HAP, TCP and collagen proteins.

Example 2: Powder Form Referring again to FIG. 1C, which is a schematic diagram of the first flow for producing the biomaterial of Example 2 of the present invention, and FIG. 1D, which is a schematic diagram of the second flow. The process of manufacturing a biomaterial in powder form is similar to the process of manufacturing a biomaterial in matrix form. As described above, the pulverized granule contains a mixture of a spongy matrix and a spongy powder. Step S23 is a step for performing filter processing. The difference in producing a biomaterial in powder form is that the powder is separated from the mixture using a filter device. Therefore, the powder form has a certain mechanical structure (the mechanical structure here means that the mechanical structure of the biomaterial is completely maintained because the present invention does not treat the biomaterial with an enzyme. In other words, not only the components such as collagen protein and hydroxyapatite are retained, but also the natural three-dimensional structure and chain structure of the material are completely retained). Furthermore, unlike the matrix form, the diameter size of the powder granules is less than 5,000 μm.

Example 3: A flake shape or an irregular shape, or a specific shape using a specific mold. See FIG. 1E, which is a schematic diagram of a first flow for producing a biomaterial according to Example 3 of the present invention. To do. The purpose is to produce a biomaterial having a specific shape from fish scales using a flake shape, an irregular lump shape, or a specific mold. This process starts from the step of decellularizing fish scales (step S30). Thereafter, a fish scale cleaning operation (step S29) is performed. The test value in the fish scale limulus test after washing should be less than 1,000 Eu / ml. Reference is made to FIG. 1F, which is a schematic diagram of a second flow for producing the biomaterial of Example 3 of the present invention. The purpose is to produce a biomaterial having a specific shape from fish scales using a flake shape, an irregular lump shape, or a specific mold. This process starts from a fish scale washing operation (step S29). The test value in the fish scale limulus test after washing should be less than 1,000 Eu / ml. Thereafter, a step (step S30) of decellularizing fish scales is performed.

  Regardless of whether the decellularization operation (step S30) is performed before washing the fish scales (step S29) or the decellularization operation (step S30) is performed after the fish scales are washed (step S29), Can also proceed to the following steps in sequence.

  Thereafter, the process proceeds to step S31, and in step S31, the fish scales are dehydrated until the water content is less than 25%. Next, in step S32, the dehydrated fish scales are crushed until the average size of the diameter of the granules is less than 5,000 μm. These granules contain a mixture of a spongy matrix and a spongy powder. Step S33 is a step of performing a filtering process, and the matrix and the powder are separated from the mixture using a filter device. In step S34, the matrix and powder are extruded. The extrusion of the matrix and the powder may be performed separately, and the extrusion may be performed after mixing the matrix and the powder. It is possible to mix the matrix and powder in different required proportions depending on the use application of the biomaterial to be manufactured, for example, biomaterial for bone repair or biomaterial for skin repair .

  There are two types of methods for producing a flake-shaped or irregular-shaped lump, or a biomaterial having a specific shape using a specific mold. The first method is a subsequent operation after the washing step (step S29). This is a direct method. As shown in FIGS. 1E and 1F, the second method is a method in which a fish scale is first washed and dehydrated before the extrusion step.

Therefore, it is an extrusion step in any case, whether it is dehydrated fish scales, undehydrated but washed fish scales, or a matrix and powdered biomaterial obtained from fish scales. To produce a biomaterial having a specific shape using a flake shape, an indeterminate lump shape, or a specific mold. First, extrusion is performed at a temperature higher than 100 g / 2.5 cm ³ at a low temperature using a whole kind of fish scales or biomaterials obtained from various kinds of fish scales. A preferred pressure is 1 kg / 2.5 cm ³ . Further, the extrusion is carried out by feeding to an extrusion mold at a high temperature of 200 ° C. (The emphasis of this step is an extrusion operation, and the extrusion temperature is merely an illustrative explanation. Low temperature (for example, −18 ° C. to 4 ° C.) Although it may be extruded at a lower temperature or at a higher temperature (for example, 30 ° C. to 60 ° C.), it should preferably not exceed 200 ° C. The specific extrusion force and extrusion time differ depending on the temperature, and the flake shape Alternatively, an indeterminate lump or a biomaterial having a specific shape using a specific mold can be obtained). Prior to the extrusion operation, the cross-linking action of the biomaterial is cross-linked by heating or adding an appropriate concentration of a chemical cross-linking agent (eg, glutaraldehyde, EDC or other commonly used chemical cross-linking agents are possible). May be. However, in biomaterials, the crosslinker can react with amines or other reactive components.

  In the decellularization process of the present invention, except that some water-soluble proteins and glucosaminoglycans are removed by washing, the original collagen protein, elastic protein fiber, and most glucosaminoglycans are still in the natural extracellular space. Kept within the quality structure. Therefore, the decellularized biomaterial can be naturally transferred to the cell, and has a considerably good biocompatibility.

  FIG. 2A is an SEM diagram in which 3T3 cells (fibroblasts 22) are cultured for 5 days with the biomaterial 20 of the present invention, and FIG. 2B is an SEM diagram in which osteoblasts 24 are cultured with the biomaterial 20 of the present invention for 5 days. is there. These figures show that the biomaterial 20 of the present invention can be used to grow fairly completely in any case, regardless of whether the cells are fibroblasts 22 or osteoblasts 24.

  FIG. 3A is a confocal microscope diagram in which 3T3 cells (fibroblasts) are cultured for 5 days with the biomaterial 20 of the present invention, and FIG. 3B is a confocal microscope in which osteoblasts are cultured for 5 days with the biomaterial 20 of the present invention. FIG. These figures show that the biomaterial 20 of the present invention can be used to grow fairly completely in any case regardless of whether it is a fibroblast or an osteoblast.

  FIG. 4 is an H & E staining diagram in which osteoblasts 24 are cultured for 5 days with the biomaterial 20 of the present invention. This figure shows that osteoblasts 24 can grow fairly completely using the biomaterial 20 of the present invention.

The foregoing is a detailed description of the invention using preferred and different embodiments, and is not intended to limit the scope of the invention. In addition, it is obvious for those skilled in the art that even if some modifications are made as appropriate, the gist and scope of the present invention are not departed.

Claims (19)

Decellularizing the fish scales and further comprising crushing into granules, wherein the granules comprise a mixture of a spongy matrix and a spongy powder;
A method for producing biomaterials from fish scales.   The method according to claim 1, further comprising extruding the spongy matrix and the spongy powder at a temperature of 200 ° C. or less to obtain a specific shape using a flaky shape or an irregular mass, or a specific mold. A method of manufacturing a biomaterial.   The method for producing a biomaterial according to claim 1, further comprising the step of dehydrating the fish scales before crushing the fish scales.   The method for producing a biomaterial according to claim 3, wherein the dehydrating step dehydrates the fish scales until the water content is less than 50%.   The method for producing a biomaterial according to claim 3, further comprising a step of washing the fish scales before the step of dewatering the fish scales.   The method for producing a biomaterial according to claim 5, wherein the step of decellularizing the fish scale is prior to the step of washing the fish scale.   The method for producing a biomaterial according to claim 3, wherein the step of decellularizing the fish scale is prior to the step of dehydrating the fish scale.   The method for producing a biomaterial according to claim 1, further comprising separating a spongy matrix and a spongy powder from the granules.   The method for producing a biomaterial according to claim 1, wherein the fish scales are pulverized until the average size of the diameter of the granules is less than 10,000 µm.   The method for producing a biomaterial according to claim 1, wherein an average size of the fish scale diameter is less than 20 cm. Decellularizing the fish scales, and further performing an extrusion process to form a flake-shaped or irregular-shaped lump, or a material having a specific shape using a specific mold,
A method for producing biomaterials from fish scales.   The method for producing a biomaterial according to claim 11, wherein the extruding step is performed at a temperature of 200 ° C. or less.   The method for producing a biomaterial according to claim 11, further comprising a step of washing the fish scales before performing the fish scale extrusion process.   The method for producing a biomaterial according to claim 13, further comprising a step of dehydrating the fish scales after completion of the washing step and before performing the fish scale extrusion process.   The method for producing a biomaterial according to claim 14, wherein the dehydrating step dehydrates the fish scales until the water content is less than 50%.   The method for producing a biomaterial according to claim 14, wherein the step of decellularizing the fish scale is prior to the step of dehydrating the fish scale.   The method for producing a biomaterial according to claim 13, wherein the step of decellularizing the fish scale is prior to the step of washing the fish scale.   The method for producing a biomaterial according to claim 11, further comprising a step of immersing the fish scale in water before performing the fish scale extrusion step. The method for producing a biomaterial according to claim 11, wherein an average size of the fish scales is less than 20 cm.